Microfluidic ultrasonic particle separators  with engineered node locations and geometries

ABSTRACT

An ultrasonic microfluidic system includes a separation channel for conveying a sample fluid containing small particles and large particles, flowing substantially parallel, adjacent to a recovery fluid, with which it is in contact. An acoustic transducer produces an ultrasound standing wave, that generates a pressure field having at least one node of minimum pressure amplitude. An acoustic extension structure is located proximate to said separation channel for positioning said acoustic node off center in said acoustic area and concentrating the large particles in said recovery fluid stream.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/524,020 filed Aug. 16, 2011entitled “microfluidic ultrasonic particle separators with engineerednode locations and geometries,” the disclosure of which is herebyincorporated by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

The United States Government has rights in this invention pursuant toContract No. DE-AC52-07NA27344 between the United States Department ofEnergy and Lawrence Livermore National Security, LLC for the operationof Lawrence Livermore National Laboratory.

BACKGROUND

1. Field of Endeavor

The present invention relates to microfluidic particle separators andmore particularly to microfluidic ultrasonic particle separators withengineered node locations and geometries.

2. State of Technology

The article, “Chip integrated strategies for acoustic separation andmanipulation of cells and particles,” by Thomas Laurell, FilipPetersson, and Andreas Nilsson in Chem. Soc. Rev., 2007, 36, 492-506,states: “Chip integrated strategies for acoustic separation andmanipulation of cells and particles,” by Thomas Laurell.” The articleincludes the state of technology information quoted below and drawingFIGS. 1A, 1B, and 1C are copies of FIGS. 5, 6 and 7 from the article.The article, “Chip integrated strategies for acoustic separation andmanipulation of cells and particles,” by Thomas Laurell, Filip Peterssonand Andreas Nilsson in Chem. Soc. Rev., 2007, 36, 492-506, isincorporated herein in its entirety for all purposes.

“FIG. 5 Schematic cross-section of separation chip utilizing the Lundmethod. The silicon separation channel is sealed by a boron silica glasslid and is actuated from below using a piezoelectric ceramic.” [FIG. 1A]

“FIG. 6 Illustrated cross-section (along the dashed line in FIG. 7) of aseparation channel showing negative w-factor particles (e.g. lipidparticles) collected in the pressure antinodes by the side walls andpositive w-factor particles (i.e. red blood cells) in the pressurenode.” [FIG. 1B]

“FIG. 7 Illustration of separation of negative w-factor particles(black—centre outlet) and positive w-factor particles (grey—sideoutlets) in 45 u design chip. [FIG. 1C]

“The Lund-method for acoustic separation of suspended particles fromtheir medium is based on a laminar flow microchannel that isultrasonically actuated from below, using a piezoelectric ceramic (FIG.5). The width of the channel is chosen to correspond to half theultrasonic wavelength, thereby creating a resonator between the sidewalls of the flow channel in which a standing wave can be formed. Theinduced standing wave is thus generated orthogonal to the incidentultrasonic wave front. As suspended particles with a positive w-factorperfuse the channel they are moved, by means of the axial PRF, towardsthe pressure nodal plane along the channel centre, while those with anegative w-factor are moved towards the anti-nodal planes close to theside walls (FIG. 6).”

“The end of the separation channel is split into three outlet channels,thus allowing the positive w-factor particles to exit through the centreoutlet and the negative w-factor particles to exit through the sideoutlets, provided that all outlet flow rates are alike (FIG. 7). Theseparation efficiency of positive and negative w-factor particles isdefined as the fraction of particles exiting through the centre and sideoutlets respectively.”

SUMMARY

Features and advantages of the present invention will become apparentfrom the following description. Applicants are providing thisdescription, which includes drawings and examples of specificembodiments, to give a broad representation of the invention. Variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this descriptionand by practice of the invention. The scope of the invention is notintended to be limited to the particular forms disclosed and theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

The present invention provides high-throughput sample processing ofbiological material. The system of the present invention separates outcell-sized particles from a background solution containing otherbiological materials (viruses, proteins, nucleic acids, etc.) in orderto allow uncontaminated analysis of either the background sample or theseparated cells. The present invention uses the vibrations of apiezoelectric transducer to produce acoustic radiation forces withinmicrofluidic channels. Whereas other investigators have demonstratedtechnologies that position a single stream of concentrated particles onthe center-line of a fluid channel, or multiple streams of particles,such that some of them are off-center, the system of the presentinvention positions a single stream of concentrated particles off-centerin the fluid channel. This is highly advantageous for achieving improvedseparation between the two sample fractions, and greater purity withineach fraction. In one embodiment, the stream of concentrated particlesis positioned off-center in the separation channel by Means ofsubdividing the channel with one or more thin acoustically transparentwalls. In another embodiment the stream of concentrated particles ispositioned off-center in the fluid flow with the aid of a polymer gelstructure positioned adjacent to the fluid channel. In theseembodiments, or in any other, the pressure field resulting from theacoustic waves can be optimized by driving the piezoelectric transducerat multiple frequencies on a single device. Additionally, in theseembodiments, or in any other, the separation channel can be routed in aserpentine fashion to pass multiple times (3, 5, etc.) through theultrasound region, thereby providing a longer residence time for thesample in the acoustic field, increasing separation efficiency.

The present invention provides an ultrasonic microfluidic system forseparating smaller particles from larger particles suspended in a samplefluid. This sample fluid flows down a separation channel, side-by-sidewith a “recovery buffer,” which is typically a fluid into which thelarger particles of interest are to be transferred. The two fluidstreams are in contact with each other, but mixing is limited only todiffusion due to the low Reynolds number of microfluidic flows. Anacoustic transducer in contact with the microfluidic chip produces anultrasound pressure field throughout these fluids. Properly tuned tomatch the geometric parameters of the channel, the acoustic transducergenerates a resonant standing wave within the fluid, creating one ormore zones of minimal pressure amplitude (acoustic nodes) toward whichparticles are driven. The forces that particles experience are dependenton particle size; therefore, the largest particles move toward the nodefastest. Positioning the node within the recovery fluid stream allowsthe largest particles to be carried out of the chip with the recoveryfluid, separating them from other sample components that remain in thesample stream. In one embodiment a second fluid channel (“bypasschannel”) is located substantially parallel to the separation channel. Awall that is thin enough to negligibly effect the transmission ofultrasound between the bypass and separation channel (“acousticallytransparent wall”) is located between the two channels. In anotherembodiment the stream of concentrated particles is positioned off-centerin the fluid channel by means of a gel positioned adjacent the fluidchannel.

The present invention enables improved operation of the acousticseparation module within the context of a Microfluidic SamplePreparation Platform that aims to purify, separate, and fractionatedifferent classes of particles (mammalian and bacterial cells, nucleicacids, viruses, etc.) from complex biological samples for both clinical(blood, urine, saliva, etc.) and environmental analysis (food, water,aerosol, etc.). These analyses have obvious applications in areas suchas biothreat detection, pathogen identification, epidemic monitoring,virology, and vaccine production, among others. The present inventioncan be used on its own or in conjunction with other sample processingsteps to prepare biological samples before they are introduced into agreat variety of commercial instruments for manipulating or analyzingbiological samples, such as DNA sequencing, PCR, flow cytometry,microorganism detection, etc.

The invention is susceptible to modifications and alternative forms.Specific embodiments are shown by way of example. It is to be understoodthat the invention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate specific embodiments of theinvention and, together with the general description of the inventiongiven above, and the detailed description of the specific embodiments,serve to explain the principles of the invention.

FIGS. 1A, 1B, and 1C (PRIOR ART) are copies of FIGS. 5, 6 and 7 from thearticle, “Chip integrated strategies for acoustic separation andmanipulation of cells and particles,” by Thomas Laurell, FilipPetersson, and Andreas Nilsson in Chem. Soc. Rev., 2007, 36, 492-506.

FIGS. 2A and 2B illustrate an embodiment of the invention wherein atransducer produces a single node.

FIGS. 3A and 3B illustrate another embodiment of the invention wherein atransducer produces two nodes.

FIGS. 4A and 4B illustrate another embodiment of the invention wherein atransducer produces two nodes, while the separation channel is designedto be narrower than the bypass channel.

FIGS. 5A and 5B illustrate another embodiment of the invention whereinthe stream of concentrated particles is positioned off-center in thefluid channel by means of a gel positioned adjacent the fluid channel.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Referring to the drawings, to the following detailed description, and toincorporated materials, detailed information about the invention isprovided including the description of specific embodiments. The detaileddescription serves to explain the principles of the invention. Theinvention is susceptible to modifications and alternative forms. Theinvention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

The present invention concerns the use of ultrasound to manipulateparticles for the purposes of separating, preparing and analyzingclinical or environmental samples containing mixtures of biologicalparticles (spores, cells, bacteria, viruses, molecules, etc.). Inparticular, when implemented within a fieldable, automated,continuous-flow system, the acoustical device is used to concentrate andfilter out the largest particles. The device is highly robust andminimally sensitive to parameter variation to interface with downstreamassays or other separation elements. The present invention can be usedon its own or in conjunction with other sample processing steps toprepare biological samples before they are introduced into a greatvariety of commercial instruments for manipulating or analyzingbiological samples, such as DNA sequencing, PCR, flow cytometry,microorganism detection, etc.

An ultrasonic particle manipulation in microfluidic device is realizedusing a piezoelectric transducer to generate acoustic standing waveswithin a microchannel. The primary acoustic radiation forces induced bythe sound waves direct particles toward the pressure-field minima(nodes) or maxima (antinodes), depending on the relative compressibilityand density between the particle and the suspending liquid. In general,if the radiation forces (which scale with particle volume) are directedtransversely to the fluid flow direction, the induced motions aresufficient to achieve continuous, high-throughput separation forparticles of diameter greater than about 2 micrometers.

EXAMPLE 1 Single Node System

Referring now to the drawings and in particular to FIG. 2A, oneembodiment of the present invention is illustrated.

The device uses multiple microfluidic channels running in parallel alongthe length of a microfluidic chip, separated by specifically-designeddistances (the “wall thickness”). One of these multiple channels servesas the separation channel and has two inlets and two outlets. Theultrasound standing-wave pressure fields are optimized to transferfocused particles out of the sample stream and into the recovery fluidwithin the recovery fluid channel. The piezoelectric transducer may bedriven at single or multiple frequencies to achieve the optimal nodeplacement depending on the channel and wall geometry. In addition,multiple small piezoelectric transducers may be arranged to producedifferent sound fields in different regions of the chip.

The present invention provides an ultrasonic microfluidic system forseparating smaller particles from larger particles suspended in a samplefluid. This sample fluid flows down a separation channel, side-by-sidewith a “recovery buffer,” which is typically a fluid into which thelarger particles of interest are to be transferred. The two fluidstreams are in contact with each other, but mixing is limited only todiffusion due to the low Reynolds number of microfluidic flows. Anacoustic transducer in contact with the microfluidic chip produces anultrasound pressure field throughout these fluids. Properly tuned tomatch the geometric parameters of the channel, the acoustic transducergenerates a resonant standing wave within the fluid, creating one ormore zones of minimal pressure amplitude (acoustic nodes) toward whichparticles are driven. The forces that particles experience are dependenton particle size; therefore, the largest particles move toward the nodefastest. Positioning the node within the recovery fluid stream allowsthe largest particles to be carried out of the chip with the recoveryfluid, separating them from other sample components that remain in thesample stream. In one embodiment a second fluid channel (“bypasschannel”) is located substantially parallel to the separation channel. Awall that is thin enough to negligibly effect the transmission ofultrasound between the bypass and separation channel (“acousticallytransparent wall”) is located between the two channels. In anotherembodiment the stream of concentrated particles is positioned off-centerin the fluid channel by means of a gel positioned adjacent the fluidchannel.

The embodiment illustrated in FIG. 2A is designated generally by thereference numeral 200. The device 200 has an “H-filter” geometry inwhich two fluids are pumped side-by-side down a microfluidic separationchannel with two inlets and two outlets. One of the two fluids containsthe sample 212, and the other fluid is a “recovery” buffer 208, which isan appropriate medium (water or buffer) into which focused particles aretransferred, while the unfocused components remain in the sample andcontinue straight through the system. Channel depth (typically 100-300micrometers), width (typ. 300-1000 micrometers), and wall thickness(typ. 10-40 micrometers) are determined for each chip based on thedesired acoustic pressure fields, and fabricated by means of standardphotolithography with anisotropic etching. The two fluids enter theseparation channel through separate inlets, and the separated samplefractions are collected at the two outlets.

The present invention provides an ultrasonic microfluidic apparatus forseparating small particles 218 from large particles 220 contained in thesample fluid 212. A sample input channel 214 is provided for conveyingthe sample fluid 212 containing small particles 218 and large particles220 toward the separation area. A recovery fluid input channel 206containing recovery fluid 208 is routed to convey the recovery fluidsubstantially parallel and adjacent the sample fluid. Within theseparation channel, the recovery fluid contacts the sample fluid 212. Abypass fluid channel 204 containing bypass fluid 202 is locatedsubstantially parallel and adjacent the separation channel 206. Anacoustically transparent wall 222 is located between the bypass channel204 and the separation channel 206. The bypass channel 204 together withthe wall 222 comprise an acoustic extension structure.

An acoustic transducer 228 in contact with the microfluidic chip 216produces an ultrasound pressure field throughout the fluids 202, 208,and 212. Properly tuned to match the geometric parameters of thechannel, the acoustic transducer 228 generates a resonant standing wavewithin the fluids 202, 208, and 212; creating one or more zones ofminimal pressure amplitude (acoustic nodes) toward which particles aredriven. The forces that particles experience are dependent on particlesize; therefore, the largest particles 220 move toward the node 224fastest. Positioning the node 224 within the recovery fluid 208 streamallows the largest particles 220 to be carried out of the chip 216 withthe recovery fluid 208, separating them from other sample componentsthat remain in the sample 212 stream.

Referring now to FIG. 2B, a cross section taken along lines 2B of FIG.2A in the direction of the arrows is shown. The body of the chip 216includes a glass cover plate 226. The body of the chip 216 and the glasscover plate 226 enclose the bypass fluid channel 204, the bypass fluid202, the recovery fluid 208, and the sample fluid 212. The acousticallytransparent wall 222 maintains the bypass fluid 208 separate from thesample 212 and recovery fluid 208 in the separation channel.

The acoustic transducer 228 produces the single acoustic node 224,designed to lie within the recovery fluid 208 stream so that therecovery fluid 208 receives the large particles 220 that areconcentrated at the acoustic node 224 causing them to be carried by therecovery fluid 208 out of the “large particle” outlet (LPO) 230.

EXAMPLE 2 Two Node System

Referring now to FIG. 3A, another embodiment of the present invention isillustrated. The device uses multiple microfluidic channels running inparallel along the length of a microfluidic chip 316, separated byspecifically-designed distances (the “wall thickness”). One of thesemultiple channels serves as the separation channel and has two inletsand two outlets. The ultrasound standing-wave pressure fields areoptimized to transfer focused particles out of the sample stream andinto the adjacent recovery fluid stream, which flow together down theseparation channel. The piezoelectric transducer 314 may be driven atsingle or multiple frequencies to achieve the optimal node placementdepending on the channel and wall geometry. In addition, multiple smallpiezoelectric transducers may be arranged to produce different soundfields in different regions of the chip.

The present invention provides an ultrasonic microfluidic system forseparating smaller particles 318 from larger particles 326 suspended ina sample fluid 312. This sample fluid 312 flows down a separationchannel 310, side-by-side with a “recovery buffer 308,” which istypically a fluid into which the larger particles 326 of interest are tobe transferred. The two fluid streams, 312 and 308, are in contact witheach other, but mixing is limited only to diffusion due to the lowReynolds number of microfluidic flows. An acoustic transducer 314 incontact with the microfluidic chip 316 produces an ultrasound pressurefield throughout these fluids. Properly tuned to match the geometricparameters of the channel, the acoustic transducer 314 generates aresonant standing wave within the fluid, creating one or more zones ofminimal pressure amplitude (acoustic nodes) toward which particles aredriven. The forces that particles experience are dependent on particlesize; therefore, the largest particles 316 move toward the nodes 324 band 324 a fastest. Positioning the node 324 b within the recovery fluid308 stream allows the largest particles 326 to be carried out of thechip 316 with the recovery fluid 308, separating them from other samplecomponents that remain in the sample 212 stream. In one embodiment asecond fluid channel (“bypass channel 304”) is located substantiallyparallel to the separation channel. A wall 322 that is thin enough tonegligibly effect the transmission of ultrasound between the bypass andseparation channel (“acoustically transparent wall 322”) is locatedbetween the two channels. In another embodiment the stream ofconcentrated particles is positioned off-center in the fluid channel bymeans of a gel positioned adjacent the fluid channel.

The present invention enables improved operation of the acousticseparation module within the context of a Microfluidic SamplePreparation Platform that aims to purify, separate, and fractionatedifferent classes of particles (mammalian and bacterial cells, nucleicacids, viruses, etc.) from complex biological samples for both clinical(blood, urine, saliva, etc.) and environmental analysis (food, water,aerosol, etc.). These analyses have obvious applications in areas suchas biothreat detection, pathogen identification, epidemic monitoring,virology, and vaccine production, among others. The present inventioncan be used on its own or in conjunction with other sample processingsteps to prepare biological samples before they are introduced into agreat variety of commercial instruments for manipulating or analyzingbiological samples, such as DNA sequencing, PCR, flow cytometry,microorganism detection, etc.

The embodiment illustrated in FIG. 3A is designated generally by thereference numeral 300. The device 300 has an “H-filter” geometry inwhich two fluids are pumped side-by-side down a microfluidic separationchannel with two inlets and two outlets. One of the two fluids is thesample 312, and the other fluid is a “recovery” buffer 308, which is anappropriate medium (water or buffer) into which focused particles aretransferred, while the unfocused components remain in the sample andcontinue straight through the system. Channel depth (typically 100-300micrometers), width (typ. 300-1000 micrometers), and wall thickness(typ. 10-40 micrometers) are determined for each chip based on thedesired acoustic pressure fields, and fabricated by means of standardphotolithography with anisotropic etching. The two fluids enter theseparation channel through separate inlets, and the separated samplefractions are collected at the two outlets.

The present invention provides an ultrasonic microfluidic apparatus forseparating small particles 318 from large particles 326 contained in thesample fluid 312. A sample input channel 310 is provided for conveyingthe sample fluid 312 containing small particles 318 and large particles326 toward the separation area. A recovery fluid input channel 306containing recovery fluid 308 is routed to convey the recovery fluidsubstantially parallel and adjacent the sample fluid. Within theseparation channel, the recovery fluid 308 contacts the sample fluid312. A bypass fluid channel 302 containing bypass fluid 304 is locatedsubstantially parallel and adjacent the separation channel. Anacoustically transparent wall 322 is located between the bypass channel302 and the separation channel. The bypass channel 302 together with thewall 322 comprise an acoustic extension structure.

An acoustic transducer 314 in contact with the microfluidic chip 316produces an ultrasound pressure field throughout these fluids. Properlytuned to match the geometric parameters of the channel, the acoustictransducer 314 generates a resonant standing wave within the fluids,creating one or more zones of minimal pressure amplitude (acousticnodes) toward which particles are driven. The forces that particlesexperience are dependent on particle size; therefore, the largestparticles 326 move toward the node fastest. Positioning the node 324 bwithin the recovery fluid 312 stream allows the largest particles 326 tobe carried out of the chip 316 with the recovery fluid 308, separatingthem from other sample components that remain in the sample 312 stream.

Referring now to FIG. 3B, a cross section taken along lines 3B of FIG.3A in the direction of the arrows is shown. The body of the chip 316includes a glass cover plate 126. The body of the chip 316 and the glasscover plate 126 enclose the bypass fluid channel 304, the bypass fluid302, the recovery fluid 308, and the sample fluid 312. The acousticallytransparent wall 322 maintains the bypass fluid 302 separate from thesample fluid 308.

The acoustic transducer 314 produces the two acoustic nodes 324 a and324 b such that the first node 32 b is located in the recovery fluid 308stream so that the recovery fluid 308 receives the large particles 326that are concentrated at the first node 324 b causing them to be carriedby the recovery fluid 308 out of the “large particle” outlet (LPO) 330.The second node 324 a is located in the bypass channel 304, and does notparticipate in the separation.

EXAMPLE 3 Alternative Fluid Channel Layout

Referring now to the drawings and in particular to FIG. 4A, anotherembodiment of the present invention is illustrated. The device usesmultiple microfluidic channels running in parallel along the length of amicrofluidic chip, separated by specifically-designed distances (the“wall thickness”). One of these multiple channels serves as theseparation channel and has two inlets and two outlets. The ultrasoundstanding-wave pressure fields are optimized to transfer focusedparticles out of the sample stream and into the recovery fluid withinthe recovery fluid channel. The piezoelectric transducer may be drivenat single or multiple frequencies to achieve the optimal node placementdepending on the channel and wall geometry. In addition, multiple smallpiezoelectric transducers may be arranged to produce different soundfields in different regions of the chip.

The embodiment illustrated in FIG. 4A is designated generally by thereference numeral 400. The device 400 has an “H-filter” geometry inwhich two fluids are pumped side-by-side down a microfluidic separationchannel with two inlets and two outlets. One of the two fluids is thesample fluid 412, and the other fluid is a “recovery” buffer 408, whichis an appropriate medium (water or buffer) into which focused particlesare transferred, while the unfocused components remain in the sample andcontinue straight through the system. Channel depth (typically 100-300μm), width (typ. 300-1000 μm), and wall thickness (typ. 10-40 μm) aredetermined for each chip based on the desired acoustic pressure fields,and fabricated by means of standard photolithography with anisotropicetching. The two fluids enter the separation channel through separateinlets, and the separated sample fractions are collected at the twooutlets.

The present invention provides an ultrasonic microfluidic apparatus forseparating small particles 418 from large particles 420 contained in thesample fluid 412. A sample input channel 410 is provided for conveyingthe sample fluid 412 containing small particles 418 and large particles420 toward the separation area. A recovery fluid input channel 406containing recovery fluid 408 is routed to convey the recovery fluidsubstantially parallel and adjacent the sample fluid. Within theseparation channel, the recovery fluid 408 contacts the sample fluid412. A bypass fluid channel 204 containing bypass fluid 204 is locatedsubstantially parallel and adjacent the separation channel. Note thatthe bypass fluid channel is a wide channel compared to the bypasschannel shown in the previously described embodiments. An acousticallytransparent wall 422 is located between the bypass channel 204 and theseparation channel 406. The bypass channel 404 together with the wall422 comprise an acoustic extension structure.

An acoustic transducer 414 in contact with the microfluidic chip 416produces an ultrasound pressure field throughout these fluids. Properlytuned to match the geometric parameters of the channel, the acoustictransducer 414 generates a resonant standing wave within the fluid,creating one or more zones of minimal pressure amplitude (acousticnodes) toward which particles are driven. The forces that particlesexperience are dependent on particle size; therefore, the largestparticles 420 move toward the node 424 b fastest. Positioning the node424 b within the recovery fluid 408 stream allows the largest particles420 to be carried out of the chip 416 with the recovery fluid 408,separating them from other sample components that remain in the sample412 stream.

Referring now to FIG. 4B, a cross section taken along lines 4B of FIG.4A in the direction of the arrows is shown. The body of the chip 416includes a glass cover plate 426. The body of the chip 416 and the glasscover plate 126 enclose the bypass fluid channel 404, the bypass fluid402, the recovery fluid 408, and the sample fluid 412. The acousticallytransparent wall 422 maintains the bypass fluid 402 separate from therecovery fluid 408.

The acoustic transducer 414 produces the acoustic nodes 424 a and 424 b.The first node 424 a is located in the recovery fluid 408 stream so thatthe recovery fluid 408 receives the large particles 420 that areconcentrated at the first acoustic node 424 a causing them to be carriedby the recovery fluid 408 out of the “large particle” outlet (LPO) 430.The second node 424 b is located in the bypass channel and does notparticipate in the separation.

EXAMPLE 4 Use of a Gel to Modify the Separation Channel Geometry

Referring now to the drawings and in particular to FIG. 5A, anotherembodiment of the invention is illustrated wherein the stream ofconcentrated particles is positioned off-center in the fluid channel bymeans of a region of hydrogel adjacent the fluid channel. In thisembodiment, rather than using a second fluid channel separated by awall, a hydrogel is immobilized (by photopolymerization or by othermeans) within the searation channel. The ultrasound standing-wavepressure fields are optimized to transfer focused particles out of thesample stream and into the recovery fluid within the recovery fluidchannel. The piezoelectric transducer may be driven at single ormultiple frequencies to achieve the optimal node placement depending onthe channel and wall geometry. In addition, multiple small piezoelectrictransducers may be arranged to produce different sound fields indifferent regions of the chip.

The embodiment illustrated in FIG. 5A is designated generally by thereference numeral 500. The device 500 has an “H-filter” geometry inwhich two fluids are pumped side-by-side down a microfluidic separationchannel with two inlets and two outlets. One of the two fluids, thesample fluid 512, contains the sample and the other fluid is a“recovery” buffer 508, which is an appropriate medium (water or buffer)into which focused particles are transferred, while the unfocusedcomponents remain in the sample and continue straight through thesystem. Channel depth (typically 100-300 micrometers), width (typ.300-1000 micrometers), and wall thickness (typ. 10-40 micrometers) aredetermined for each chip based on the desired acoustic pressure fields,and fabricated by means of standard photolithography with anisotropicetching. The two fluids enter the separation channel through separateinlets, and the separated sample fractions are collected at the twooutlets.

The present invention provides an ultrasonic microfluidic apparatus forseparating small particles 518 from large particles 520 contained in thesample fluid 512. A sample input channel 510 is provided for conveyingthe sample fluid 512 containing small particles 518 and large particles520 toward the separation area. A recovery fluid input channel 506containing recovery fluid 508 is routed to convey the recovery fluidsubstantially parallel and adjacent the sample fluid. Within theseparation channel, the recovery fluid contacts the sample fluid 512. Agel 502 is located substantially parallel and adjacent the separationchannel 506. The gel 502 comprises an acoustic extension structure.

An acoustic transducer 514 in contact with the microfluidic chip 516produces an ultrasound pressure field throughout these fluids. Properlytuned to match the geometric parameters of the channel, the acoustictransducer 514 generates a resonant standing wave within the fluid,creating one or more zones of minimal pressure amplitude (acousticnodes) toward which particles are driven. The forces that particlesexperience are dependent on particle size; therefore, the largestparticles 420 move toward the node 424 fastest. Positioning the node 524within the recovery fluid 508 stream allows the largest particles 520 tobe carried out of the chip 516 with the recovery fluid 508, separatingthem from other sample components that remain in the sample stream.

Referring now to FIG. 5B, a cross section taken along lines 5B of FIG.5A in the direction of the arrows is shown. The body of the chip 516includes a glass cover plate 126. The body of the chip 516 and the glasscover plate 126 enclose the gel 528, the recovery fluid 508, and thesample fluid 512. The acoustic transducer 514 produces the acoustic node524 in the recovery fluid stream 506 so that the recovery fluid 508receives the large particles 520 that are concentrated at the acousticnode 524 causing them to be carried by the recovery fluid 508 out of the“large particle” outlet (LPO) 530.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and have been described in detail herein.However, it should be understood that the invention is not intended tobe limited to the particular forms disclosed. Rather, the invention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the followingappended claims.

1. An ultrasonic microfluidic apparatus for separating small particlesfrom large particles contained in a sample fluid, comprising: a sampleinput channel for channeling the sample fluid containing the smallparticles and the large particles; a recovery fluid input channelcontaining recovery fluid, routing said recovery fluid to flowsubstantially parallel and adjacent to the sample fluid, wherein saidrecovery fluid contacts the sample fluid; an acoustic transducer thatproduces an acoustic standing wave, that generates a pressure fieldhaving at least one node of minimum sound pressure amplitude; and anacoustic extension structure located proximate the sample fluid and saidrecovery fluid that positions said at least one acoustic node in saidrecovery fluid concentrating the large particles in said recovery fluid.2. The ultrasonic microfluidic apparatus for separating small particlesfrom large particles contained in a sample fluid of claim 1 wherein saidacoustic extension structure includes a bypass fluid channel containingbypass fluid, said bypass fluid channel located substantially paralleland adjacent the sample fluid and said recovery fluid; and anacoustically transparent wall between said bypass channel and the samplefluid and said recovery fluid.
 3. The ultrasonic microfluidic apparatusfor separating small particles from large particles contained in asample fluid of claim 1 wherein said acoustic extension structureincludes a gel located proximate the sample fluid and said recoveryfluid positioning said at least one acoustic node in said recovery fluidconcentrating the large particles in said recovery fluid.
 4. Theultrasonic microfluidic apparatus for separating small particles fromlarge particles contained in a sample fluid of claim 1 wherein thesample fluid and said recovery fluid are positioned to form a separationchannel, and wherein said at least one node of minimum sound pressureamplitude is positioned off center in said separation channelconcentrating the large particles in said recovery fluid.
 5. Theultrasonic microfluidic apparatus for separating small particles fromlarge particles contained in a sample fluid of claim 1 wherein saidacoustic standing wave generates a pressure field having a single nodeof minimum sound pressure amplitude concentrating the large particles insaid recovery fluid stream.
 6. The ultrasonic microfluidic apparatus forseparating small particles from large particles contained in a samplefluid of claim 1 wherein said acoustic standing wave generates two ormore acoustic nodes of minimum sound pressure amplitude concentratingthe large particles in said recovery fluid stream.
 7. An ultrasonicmicrofluidic apparatus for separating small particles from largeparticles contained in a sample fluid, comprising: a sample fluid streamcontaining the small particles and the large particles; a recovery fluidstream located substantially parallel and adjacent to said sample fluidstream, wherein said recovery fluid stream contacts said sample fluidstream; an acoustic transducer that produces an acoustic standing wave,that generates a pressure field having at least one node of minimumsound pressure amplitude; and an acoustic extension structure locatedproximate said sample fluid stream and said recovery fluid stream thatpositions said at least one acoustic node in said recovery fluid streamconcentrating the large particles in said recovery fluid stream.
 8. Theultrasonic microfluidic apparatus for separating small particles fromlarge particles contained in a sample fluid of claim 7 wherein saidacoustic extension structure includes a bypass fluid channel containingbypass fluid, said bypass fluid channel located substantially paralleland adjacent said sample fluid stream and said recovery fluid stream;and an acoustically transparent wall between said bypass channel andsaid sample fluid stream and said recovery fluid stream.
 9. Theultrasonic microfluidic apparatus for separating small particles fromlarge particles contained in a sample fluid of claim 7 wherein saidacoustic extension structure includes a gel located proximate saidsample fluid stream and said recovery fluid stream positioning said atleast one acoustic node in said recovery fluid stream concentrating thelarge particles in said recovery fluid stream.
 10. The ultrasonicmicrofluidic apparatus for separating small particles from largeparticles contained in a sample fluid of claim 7 wherein said samplefluid stream and said recovery fluid stream are positioned to form aseparation channel, and wherein said at least one node of minimum soundpressure amplitude is positioned off center in said separation channelconcentrating the large particles in said recovery fluid stream.
 11. Theultrasonic microfluidic apparatus for separating small particles fromlarge particles contained in a sample fluid of claim 7 wherein saidacoustic standing wave generates a pressure field having a single nodeof minimum sound pressure amplitude concentrating the large particles insaid recovery fluid stream.
 12. The ultrasonic microfluidic apparatusfor separating small particles from large particles contained in asample fluid of claim 7 wherein said acoustic standing wave generatestwo or more acoustic nodes of minimum sound pressure amplitudeconcentrating the large particles in said recovery fluid stream.
 13. Anultrasonic microfluidic apparatus for separating small particles fromlarge particles contained in a sample fluid, comprising: means forproviding a sample channel for channeling the sample fluid containingthe small particles and the large particles; means for providing arecovery fluid channel routed to convey recovery fluid substantiallyparallel and adjacent said sample stream wherein said recovery fluidcontacts the sample fluid; means for providing an acoustic extensionstructure proximate said sample channel and said recovery fluid channel;means for using an acoustic transducer for producing an ultrasoundpressure field having at least one node of minimum pressure amplitude,said pressure field encompassing said separation channel, and saidacoustic extension structure; wherein said acoustic transducer positionssaid at least one node off center in said separation channelconcentrating the large particles in said recovery fluid stream.
 14. Theultrasonic microfluidic apparatus for separating small particles fromlarge particles contained in a sample fluid of claim 13 wherein saidmeans for locating an acoustic extension structure proximate said samplechannel and said recovery fluid channel comprises means for locating abypass fluid channel containing bypass fluid substantially parallel andadjacent said separation channel and means for locating an acousticallytransparent wall between said bypass channel and said separationchannel.
 15. The ultrasonic microfluidic apparatus for separating smallparticles from large particles contained in a sample fluid of claim 13wherein said means for locating an acoustic extension structureproximate said separation channel comprises means for locating a gelregion proximate said sample channel and said recovery channelpositioning said at least one acoustic node off center in saidseparation channel concentrating the large particles in said recoveryfluid stream.
 16. An ultrasonic microfluidic method for separating smallparticles from large particles contained in a sample fluid, comprisingthe steps of: providing a sample channel for channeling the sample fluidcontaining the small particles and the large particles; routing arecovery fluid channel to flow recovery fluid substantially parallel andadjacent said sample fluid, wherein said recovery fluid contacts thesample fluid thereby creating a separation channel; locating an acousticextension structure proximate said separation channel; and using anacoustic transducer for producing an ultrasound standing wave thatgenerates a pressure field having at least one node of minimum pressureamplitude, said pressure field encompassing said separation channel, andsaid acoustic extension structure; wherein said acoustic transducerpositions at least one acoustic node off center in said separationchannel concentrating the large particles in said recovery fluid stream.17. The ultrasonic microfluidic method for separating small particlesfrom large particles contained in a sample fluid of claim 16 whereinsaid step of locating an acoustic extension unit proximate said samplechannel and said recovery fluid channel comprises locating a bypassfluid channel containing bypass fluid substantially parallel andadjacent said recovery fluid channel and locating an acousticallytransparent wall between said bypass channel and said recovery fluidchannel.
 18. The ultrasonic microfluidic method for separating smallparticles from large particles contained in a sample fluid of claim 16wherein said step of locating an acoustic extension unit proximate saidsample channel and said recovery fluid channel comprises locating a gelunit proximate said sample channel and said recovery channel positioningsaid at least one acoustic node off center in said acoustic areaconcentrating the large particles in said recovery fluid channel. 19.The ultrasonic microfluidic method for separating small particles fromlarge particles contained in a sample fluid of claim 16 wherein saidstep of using an acoustic transducer for producing an acoustic areacomprises using an acoustic transducer for producing a single acousticnode that concentrates the large particles in said recovery fluidchannel.
 20. The ultrasonic microfluidic method for separating smallparticles from large particles contained in a sample fluid of claim 16wherein said step of using an acoustic transducer for producing anacoustic area comprises using an acoustic transducer for producing atleast two acoustic nodes that concentrate the large particles in saidrecovery fluid channel.
 21. An ultrasonic microfluidic method forseparating small particles from large particles contained in a samplefluid, comprising the steps of: providing a sample fluid streamcontaining the small particles and the large particles; providing arecovery fluid stream located substantially parallel and adjacent tosaid sample fluid stream, wherein said recovery fluid stream contactssaid sample fluid stream; providing an acoustic transducer that producesan acoustic standing wave, that generates a pressure field having atleast one node of minimum sound pressure amplitude; and providing anacoustic extension structure located proximate said sample fluid streamand said recovery fluid stream that positions said at least one acousticnode in said recovery fluid stream concentrating the large particles insaid recovery fluid stream.
 22. The ultrasonic microfluidic method forseparating small particles from large particles contained in a samplefluid of claim 21 wherein said step of providing an acoustic extensionstructure located proximate said sample fluid stream and said recoveryfluid stream includes providing a bypass fluid stream containing bypassfluid substantially parallel and adjacent said recovery fluid stream andlocating an acoustically transparent wall between said bypass fluidstream and said recovery fluid stream.
 23. The ultrasonic microfluidicmethod for separating small particles from large particles contained ina sample fluid of claim 21 wherein said step of providing an acousticextension structure located proximate said sample fluid stream and saidrecovery fluid stream includes providing a gel proximate said recoveryfluid stream.